Bottom Line:
Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells.High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin.In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

ABSTRACTResistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-κB signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-κB was hyperactivated in in vitro models of OXA-acquired resistance but was attenuated by the addition of Curcumin, a non-toxic NF-κB inhibitor. The concomitant combination of Curcumin + OXA was more effective and synergistic in cell lines with acquired resistance to OXA, leading to the reversion of their resistant phenotype, through the inhibition of the NF-κB signalling cascade. Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells. Moreover, CXCL8 and CXCL1 gene silencing made resistant cells more sensitive to OXA through the inhibition of the Akt/NF-κB pathway. High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin. In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

Mentions:
Previous results from our group suggested an important role for the NF-κB pathway in OXA resistance acquisition in in vitro models12. In the present work, we tried to demonstrate this hypothesis by first investigating the NF-κB basal status in 3 CRC Cell lines (HT29, LoVo and DLD1) and their corresponding OXA-resistant derived sub-lines (HTOXAR3, LoVOXAR3 and DLDOXAR3, respectively). As shown in Fig. 1 all the cell lines had constitutively phosphorylated p65 subunit at Ser536 which is considered an indicator of this pathway’s activity9. However, we observed an increase in this phosphorylation in HTOXAR3 (Fig. 1a,b) and DLDOXAR3 (Fig. 1a,d) as compared with their parental cell lines. Besides this, increased levels of phosphorylated IκBα at Ser32/36 residues, indicating degradation of this NF-κB inhibitor by the proteasome and p65 nuclear translocation, were also found in all three resistant cell lines in comparison with their respective parental ones. Taking into account that Survivin, Bcl-2 and Cyclin D1 have all been shown to be transcriptionally regulated by NF-κB6, we examined the basal expression levels of these proteins by western blot. We observed that all of them were significantly overexpressed in HTOXAR3 as compared to HT29 cells (Fig. 1a,b). In the case of the LoVo/LoVOXAR3 pair we observed a significant increase in Bcl-2 and Cyclin D1 protein expression in the resistant cell line (Fig. 1a,c) while only Survivin protein expression showed statistically significant differences between DLDOXAR3 and DLD1 (Fig. 1a,d). The Serine/threonine kinase Akt plays a critical role in proliferation and cell survival and its implication in the activation of anti-apoptotic mechanisms such as those driven by NF-κB is well documented2829. In agreement with this, in our previous study we reported increased levels of AKT1 mRNA in OXA-resistant cell lines12. Here we also demonstrate a significantly increased phosphorylation of Akt at Ser473 in HTOXAR3 as compared to HT29 cells (Fig. 1a,b) suggesting a possible role for Akt in the NF-κB pathway activation in the HTOXAR3 cell line.

Mentions:
Previous results from our group suggested an important role for the NF-κB pathway in OXA resistance acquisition in in vitro models12. In the present work, we tried to demonstrate this hypothesis by first investigating the NF-κB basal status in 3 CRC Cell lines (HT29, LoVo and DLD1) and their corresponding OXA-resistant derived sub-lines (HTOXAR3, LoVOXAR3 and DLDOXAR3, respectively). As shown in Fig. 1 all the cell lines had constitutively phosphorylated p65 subunit at Ser536 which is considered an indicator of this pathway’s activity9. However, we observed an increase in this phosphorylation in HTOXAR3 (Fig. 1a,b) and DLDOXAR3 (Fig. 1a,d) as compared with their parental cell lines. Besides this, increased levels of phosphorylated IκBα at Ser32/36 residues, indicating degradation of this NF-κB inhibitor by the proteasome and p65 nuclear translocation, were also found in all three resistant cell lines in comparison with their respective parental ones. Taking into account that Survivin, Bcl-2 and Cyclin D1 have all been shown to be transcriptionally regulated by NF-κB6, we examined the basal expression levels of these proteins by western blot. We observed that all of them were significantly overexpressed in HTOXAR3 as compared to HT29 cells (Fig. 1a,b). In the case of the LoVo/LoVOXAR3 pair we observed a significant increase in Bcl-2 and Cyclin D1 protein expression in the resistant cell line (Fig. 1a,c) while only Survivin protein expression showed statistically significant differences between DLDOXAR3 and DLD1 (Fig. 1a,d). The Serine/threonine kinase Akt plays a critical role in proliferation and cell survival and its implication in the activation of anti-apoptotic mechanisms such as those driven by NF-κB is well documented2829. In agreement with this, in our previous study we reported increased levels of AKT1 mRNA in OXA-resistant cell lines12. Here we also demonstrate a significantly increased phosphorylation of Akt at Ser473 in HTOXAR3 as compared to HT29 cells (Fig. 1a,b) suggesting a possible role for Akt in the NF-κB pathway activation in the HTOXAR3 cell line.

Bottom Line:
Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells.High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin.In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

ABSTRACTResistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-κB signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-κB was hyperactivated in in vitro models of OXA-acquired resistance but was attenuated by the addition of Curcumin, a non-toxic NF-κB inhibitor. The concomitant combination of Curcumin + OXA was more effective and synergistic in cell lines with acquired resistance to OXA, leading to the reversion of their resistant phenotype, through the inhibition of the NF-κB signalling cascade. Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells. Moreover, CXCL8 and CXCL1 gene silencing made resistant cells more sensitive to OXA through the inhibition of the Akt/NF-κB pathway. High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin. In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.